Project Summary/Abstract: We now appreciate that Cystic Fibrosis (CF) is caused by multiple variants defined by CFTR2 comprising >300 clinically validated variants contributing to disease. Deletion of Phe 508 from the first nucleotide binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) protein is the prominent mutation found in ~90% patients, a residue critical for both intra- domain and inter-domain contacts controlling the intrinsic thermodynamic stability of the CFTR fold and channel function. Folding of CFTR and its function is determined not only by its primary sequence, but by an extensive proteostasis network that chaperones the dynamic protein fold throughout it?s lifespan. In addition to an improved understanding of the roles of proteostasis and interaction networks during the previous funding period by the Balch laboratory, the Riordan group has made major advances in addressing the structural elements of CFTR responsible for its marginal thermodynamic stability that make the fold susceptible to complete destabilization by single amino acid changes. The unifying hypothesis from Balch and Riordan underpinning this proposal is that variants impacting the ?functional? structure of CFTR in the cell are manifested as networking problems within the dynamic protein conformational organization of CFTR (cis interactions) and between this dynamic structural network and components of proteostasis network in trans. Our objective is to understand the relationship(s) between these cis and trans networks in depth by focusing on the properties of the fold found in vivo (Balch) and relating these biological properties of the physiololgic fold to biochemical, biophysical and structural features defined in vitro (Riordan). Unique to our hypothesis is the postulate that proteostasis biology can be modified to ?repair? the function of CFTR folding mutants by impacting their stability and functional dynamics. These issues will be addressed in two Aims. Aim 1 (Balch and Riordan) will jointly focus on understanding key proteostasis components contributing to loss and correction of F508 function in the NBD1 domain in vivo (Balch) in relation to its conformational stability in vitro (Riordan). Balch will investigate the role of factors influencing mRNA stability, translation and early folding events that we hypothesize are critical to management of the structural defects directing export from the endoplasmic reticulum (ER) for downstream function. The Riordan laboratory exploit major advances made in the expression and purification of high quality wild-type (WT) and variant CFTR protein suitable to understand these NBD1 defined events at the biochemical, biophysical and structural levels in vitro in the context of the full- length protein. Aim 2 will expand Aim1 to focus on additional rare CFTR2 variants that tune the fold of NBD1 to understand the differential impact of proteostasis in the global management of NBD1 with the hypothesis that each variant provides a unique view into intermediate folding states responsible for function. Integration of the long-term Balch and Riordan efforts will now provide an unprecedented understanding of the CFTR folding landscape.